The present invention relates to a process for preparing an immobilized enzyme showing a high activity with a less loss in the enzyme activity, which is used for hydrolysis of fats and oils, ester-exchange of fats and oils, and esterification of aliphatic acids and alcohols. The term of xe2x80x9cfats and oilsxe2x80x9d means an inclusion of a fat, an oil, a lard, a grease and so on.
In hydrolyzing fats and oils by a lipolytic (or fat and/or oil-decomposing) enzyme, an immobilized enzyme prepared by immobilizing a lipolytic enzyme onto an inorganic or organic carrier is used for efficient use of the enzyme. To raise the absorptivity of the enzyme onto a carrier and to improve an enzyme activity, various studies have been made, and for example, JP-A 9-257 discloses a process for producing an immobilized enzyme carrier prepared by immobilizing a lipase onto an inorganic carrier treated with a silane coupling agent having a special functional group, washing and drying it, and impregnating it with an aliphatic acid. Even by this method, however, the amount of the adsorbed enzyme and the enzyme activity remain still insufficient.
Further, an immobilized enzyme prepared by immobilizing a lipolytic enzyme called lipase onto a carrier is used as an enzyme mainly for use in reactions for the object of ester-exchanging (ester-interchanging or transesterifying) fats and oils and esterifying aliphatic acids and alcohols. These reactions are advantageously conducted at concentrations of water as low as possible (1000 ppm or less) to inhibit hydrolysis, and thus the immobilized enzyme is forcibly dried to give only several % water content in the carrier, since the immobilized enzyme is prepared.
However, the adsorbed enzyme tends to inactivate in the step of drying the immobilized enzyme, and there are many cases where the enzyme does not exhibit the maximum activity upon adsorption and when the enzyme exhibits activity thereof actually.
JP-A 62-134090 describes that after immobilization, the immobilized enzyme is dried under contact with aliphatic acid derivatives thereby raising its activity expression, but this method is neither practical nor efficient because expensive facilities are necessary for drying the immobilized enzyme and furthermore it is complicated to set up conditions etc. for slow drying.
Under these circumstances, it is desired that a lipolytic enzyme is prepared so as to exhibit its activity expression sufficiently and to prevent the enzyme from being left (or removed) or inactivated, whereby the amount of the enzyme used for lipolysis is reduced and the esterification reaction is promoted.
To solve this problem, it is desirable that a larger amount of a lipolytic enzyme is adsorbed at adsorption step so as to express high activity and that an atmosphere for promoting the reaction is created around the immobilized enzyme. The present invention relates to a process for preparing an immobilized enzyme for a lipolysis (or decomposition of fats and oils), which comprises absorbing and immobilizing the enzyme onto a porous, anion-exchanging resin as an immobilizing carrier, and which is treated with fats and oils, and the problem described above was thereby solved.
As a result of eager study by the present inventors to solve this problem, they found that it is necessary to confer a stable state on the enzyme adsorbed onto the carrier, for which it is effective to bring a reaction substrate. (or reactant) into contact with the enzyme rapidly after immobilization.
That is, the present invention relates to a process for esterification reaction, which comprises immobilizing a lipolytic enzyme on a carrier for immobilization by adsorption and, without drying, directly bringing the immobilized enzyme into contact with its substrate.
That is, in the present invention, the lipolytic enzyme in a un-dried state after immobilization is brought into contact with its substrate there by providing the immobilized enzyme with a higher degree of adsorption and a higher activity.
The invention provides a process for preparing an immobilized enzyme, which comprises the steps of:
immobilizing a lipolytic enzyme on a porous, anion-exchanging resin for a carrier by adsorption and,
without drying, treating the immobilized enzyme with fats and oils or a derivative of fats and oils.
The process may preferably further comprise the step of treating the carrier""s resin with a lypophylic (or fat and/or oil-solving) aliphatic acid or a derivative of a lypophylic aliphatic acid in advance to the immobilization step.
The immobilized enzyme may be treated with fats and oils and the obtained immobilized enzyme is usable for hydrolysis.
Alternatively the immobilized enzyme may be treated with the derivative of fats and oils and the obtained immobilized enzyme is usable for esterification.
The used enzyme is preferably lipase.
The invention provides also use of the immobilized enzyme as defined above for hydrolysis or esterification of fats and oils or a derivative of fats and oils, for example hydrolysis of fats and oils, esterification of derivatives of fats and oils such as partial glycerides, glycerol and an aliphatic acid.
In addition, the invention provides a process for esterifying reaction substrates, which comprises the steps of:
immobilizing a lipolytic enzyme on a porous, anion-exchanging resin for a carrier by adsorption and,
without drying, bringing the immobilized enzyme into contact with the reaction substrates.
The invention moreover provides a process for hydrolysis of reaction substrates, which comprises the steps of:
immobilizing a lipolytic enzyme on a porous, anion-exchanging resin for a carrier by adsorption and,
without drying, treating the immobilized enzyme with reaction substrates and
hydrolyzing the reaction substrates.
In esterification and hydrolysis, the reaction substrates may be fats and oils, such as triglycerides, or a derivative of fats and oils. The derivative of fats and oils may be an aliphatic acid, glycerol or partial glycerides such as monoglycerides and diglycerides.
According to the invention, the immobilized enzyme can be treated with the reaction substrates for hydrolysis or esterification, immediately being subject to the reaction. Alternatively, the immobilized enzyme can be stored after treatment with fats and oils or a derivative of fats and oils.
The carrier used in the present invention is preferably a porous, anion-exchanging resin. The particle diameter of the resin is desirably 400 to 1000 xcexcm, and the diameter of its pore is desirably 100 to 1500 xc3x85.
The resin materials include phenol formaldehyde based, polystyrene based, acrylamide based, divinyl benzene based. In particular, phenol formaldehyde-based resin (e.g. tradename: Duolite A-568) is desirable. Its pores give a large surface area for adsorption of the enzyme to obtain a larger amount for adsorption.
In the present invention, the carrier is treated preferably with a lypophylic aliphatic acid or a lypophylic aliphatic acid derivative for pre-treatment before immobilization, thus creating a state of adsorption to exhibit a high activity. The lypophylic aliphatic acid or lypophylic aliphatic acid derivative used has preferably 8 to 18 carbon atoms. For example, said aliphatic acid includes linear and saturated aliphatic acids such as capric acid, lauric acid and myristic acid, unsaturated aliphatic acids such as oleic acid and linoleic acid, hydroxy aliphatic acids such as ricinoleic acid, or branched aliphatic acids such as isostearic acid. The aliphatic acid derivative includes esters between C8 to C18 aliphatic acids and compounds having a hydroxyl group, and examples thereof include mono-alcohol monohydric alcohol or monovalent alcohol) esters, polyhydric alcohol polyol or polyvalent alcohol) esters, phospholipids, or derivatives of these esters to which ethylene oxide has been added. The mono-alcohol esters include methyl ester, ethyl ester, and the polyvalent alcohol esters include monoglyceride, diglyceride and derivatives thereof, or polyglycerol fatty acid esters, sorbitan fatty acid esters, sucrose fatty esters.
It is desirable for the process that any of these aliphatic acids and derivatives thereof is in the state of liquid at ordinary temperatures. These may be used alone, or these are combined to bring about further effects. It is considered that these derivatives are chemically decomposed in an aqueous catalyst or hydrolyzed with a lipolytic enzyme, to form aliphatic acids.
To bring these lypophylic aliphatic acids and derivatives thereof into contact with the porous anion-exchange resin, they may be added directly as such to water or an organic solvent, or to improve dispersibility, the lypophylic aliphatic acids or derivatives thereof are once dispersed and dissolved in a solvent and then they may be added to the porous anion-exchange resin dispersed in water. The organic solvent used in this step includes chloroform, hexane, ethanol. The ratio of the lypophylic aliphatic acid or the derivative thereof to the porous anion-exchange resin is preferable to be 0.01 to 1 part by weight, and particularly preferable to be 0.05 to 0.5 part by weight, of the lypophylic aliphatic acid or the derivative thereof as compared with 1 part (dry weight basis) by weight of the porous, anion exchange resin. The temperature for contact is 0 to 100xc2x0 C., preferably 20 to 60xc2x0 C. The time for contact may be about 5 minutes to about 5 hours. After this treatment, the resin is recovered by filtration and then may be dried at this time. The temperature for drying is preferably room temperature to 100xc2x0 C., and drying under reduced pressure may be conducted.
The lipolytic enzyme used in the present invention includes lipases derived (or originated) from microorganisms (microbes or germs) of the genera Rizopus, Aspergillus, Chromobacterium, Mucor, Pseudomonas, Geotrichum, Penicillium and Candida as well as animal lipases such as pancreatic lipase. To obtain aliphatic acids at high degrees of decomposition or to obtain triglycerides at high degrees of esterification, a lipase (random type) having no position selectivity is preferable, and the enzyme derived from the microorganism is preferably selected from the genera Pseudomonas and Candida.
To obtain a partial glyceride such as monoglyceride and diglyceride at high degrees of esterification, a lipase having position selectivity is preferable.
The temperature for conducting an immobilization is well 0 to 60xc2x0 C., preferably 5 to 40xc2x0 C., because of no arising inactivation of an enzyme, but the temperature can be selected depending on the characteristics of the enzyme used. The pH of the enzyme solution is well in the range as far as the enzyme is not denatured. The pH 3 to 9 is desirable. The pH can also be determined similarly to the temperature, depending on the characteristics of the enzyme. The buffer for maintaining the pH includes, but is not limited to, acetic acid-based buffer, phosphoric acid-based buffer and Tris-HCl-based buffer.
In the method of immobilization according to the present invention, the concentration of the enzyme in the enzyme solution is desirable to be the solubility of the enzyme or below and to be sufficient concentration, in respect of immobilization efficiency. If necessary, insoluble enzymes were removed by centrifugation, and then a supernatant can be used. The ratio of the enzyme to the carrier for immobilization is preferable to be 0.05 to 10 part by weight, and particularly preferable to be 0.1 to 5 part by weight, of the enzyme as compared with 1 part by weight of the carrier for immobilization.
To bring the enzyme into contact with the carrier treated as described above, it is possible to use a method of dispersing and stirring a carrier in an enzyme solution or a method of introducing a carrier into a packing tower (or packing column) such as column etc. and circulating an enzyme solution through it using a pump and so on. Any method thereof may be used.
The process up to this step in preparation of the immobilized enzyme for hydrolysis of fats and oils may be the same as in preparation of the immobilized enzyme for esterification reaction.
Hereinafter, preferable embodiments for preparing the immobilized enzyme for hydrolysis of fats and oils are described.
The reaction substrate, which is used in the present invention and which is made to treat the immobilized enzyme after immobilization, includes the fats and oils such as rapeseed oil, soybean oil, corn oil, olive oil, tallow, fish oil. Although they are not limited, fats and oils actually hydrolyzed are desirably used.
To bring the substrate into contact with the immobilized enzyme after immobilization, the immobilized enzyme is recovered by filtration from the enzyme solution after immobilization, then excess water content is removed, and without drying, the immobilized enzyme is brought into contact with fats and oils as the substrate. The water content in the immobilized enzyme, though being varied depending on the type of carriers used, is 20% or more by weight and preferably in the range of 40 to 60% by weight. The immobilized enzyme may be introduced into a packing vessel such as column to circulate fats and oils through the packing vessel with e.g. a pump, or the immobilized enzyme may be dispersed in fats and oils. The temperature for contact is preferable to be ordinary temperature to 60xc2x0 C., and this temperature can be selected depending on the characteristics of the enzyme. Further, the time for contact may be about 2 hours to about 24 hours. After this contact is finished, it is filtered to recover the immobilized enzyme. By this operation, the reaction site of the immobilized enzyme is considered to become suitable for hydrolysis. The immobilized enzyme after subjected to this treatment is good in storage stability. This is considered due to the stabilization of lipase by fats and oils.
Hereinafter, preferable embodiments for preparing the immobilized enzyme for esterification reaction are described.
The substrate includes C2 to C22 aliphatic acids. These may be either saturated or unsaturated, or may contain a linear chain besides a branched chain and/or a conjugated double bond etc. Further, the substrate may contain structural isomers thereof and is not particularly limited. For preparation of esters having a single aliphatic acid component, partial glycerides and/or triglycerides, these aliphatic acids can be used alone or may be used as a mixture of two or more type thereof. Further, the aliphatic acids may be used as completely or partially decomposed from one or more vegetable oils and/or animal fats. On the other hand, the alcohols include C1 to C22 monohydric alcohols and dihydric or more-hydric alcohols. As the substrate, the aliphatic acids and alcohols described above are combined such that a desired esterified product can be produced, and the substrate is not limited to specific compounds.
The immobilized enzyme after immobilized by adsorption is deprived (or removed) of water sufficiently by a physical method and then brought into contact with the substrate to effect the esterification reaction. The water content in the immobilized enzyme, though being varied depending on the type of carriers used, is usually 20% or more by weight and preferably in the range of 40 to 60% by weight.
In the case of the esterification reaction, the reaction is conducted by shift under dehydration, and therefore, while the reaction is conducted, excess water content remaining in the immobilized enzyme can simultaneously be removed by use of a dehydration system. After the lipolytic enzyme is immobilized by adsorption onto a carrier for immobilization, the initial esterification reaction is conducted by directly bringing the immobilized enzyme without drying into contact with the substrate, and removal of this excess water content in this initial esterification reaction requires extra reaction time but can be effected in a considerably shorter time than the time for conventionally conducted drying of the immobilized enzyme. Further, the composition and qualities of a product produced in this initial reaction are in no way inferior to those of a product produced in the second or later reaction. In this initial esterification reaction, the time elapsed until a water content being enough for enzyme to exhibit its activity, though being varied depending on the amount of the immobilized enzyme and on the ability of the dehydration system used, is approximately 1 hour or so. Because excess water content was removed in the initial reaction, the time required for removal of water in the initial reaction is not necessary in the second or later reaction in which the immobilized enzyme for esterification reaction can be obtained.
For the method for esterification reaction, it is possible to use any methods known in the public art, such as dehydration under reduced pressure, glycerol dehydration, and dehydration using a dehydrating agent such as molecular sieves. Further, the immobilized enzyme maybe used in a stirring reactor, a packed column reactor and a fluidized bed reactor.
According to the present invention, the maximum activity of the adsorbed enzyme is brought about and the esterification reaction can be conducted efficiently and stably for a long period of time by conferring a stable state on the immobilized enzyme and by maximally preventing the enzyme from being inactivated due to drying.